Vitamin A deficiency is a global health problem affecting close to 200 million children. Vitamin A can not be synthesized de novo and it is essential for growth and development;deficiencies manifest as xerophthalmia, blindness, and increased mortality. Plant-derived carotenoids are converted in humans to vitamin A. An approach to alleviating worldwide deficiency is to improve provitamin A carotenoid levels in food staples such as corn, wheat, and rice by metabolic engineering and/or in combination with marker-assisted selection. Preliminary success with metabolic engineering of the pathway in plants points to the potential of this approach. Unexpected products in such transgenic plants, however, suggest that the technology is limited by current deficiencies in understanding of endogenous gene expression. Rational metabolic engineering strategies must take into account the regulation of the endogenous pathway which is not yet completely understood. We propose that the relative accumulation of provitamin A carotenoids is mediated by control of transcript levels for the biosynthetic and degradative enzymes;and that modification (enhancement or repression) of transcript levels in corn endosperm can lead to increased levels of provitamin A carotenoids relative to other non-provitamin A carotenoids. NIH SCORE funding has allowed us to begin to develop tools and research which are most immediately applicable to two of the most important food crops worldwide--corn and rice. We discovered that several pathway enzymes are encoded by small gene families, which raises the question: what is the contribution of gene family members to carotenoid biosynthesis and accumulation in different tissues, developmental stages, and plastid membrane localization? We will continue our NIH-SCORE supported work to conduct a comprehensive investigation of expression of endogenous genes which encode enzymes of the carotenoid pathway and the related isoprenoid biosynthetic pathway which provides substrates for carotenoid biosynthesis. We will also investigate genes encoding enzymes that degrade or modify carotenoids and thus diminish provitamin A content. The proposed research will be divided into two areas: (1) transcript profiling related to carotenoid synthesis/ degradation and correlation with carotenoid content/ composition and (2) characterization of the carotene ring hydroxylases as they impact conversion of provitamin A carotenes to non-provitamin A xanthophylls. Relevance: Vitamin A deficiency is a global health problem affecting close to 200 million children. To alleviate deficiency worldwide, crop plants can be improved for provitamin A carotenoid content, the goal of which requires breeding tools and basic research on how carotenoid composition is regulated in food crops, beginning with corn (maize) and rice as models.